Introduction and Aims: Estrogens have a positive impact on the auditory function. Indeed, estradiol (E2) treatment in animals protects against acoustic trauma and aminoglycoside toxicity and its depletion ... [more ▼]

Introduction and Aims: Estrogens have a positive impact on the auditory function. Indeed, estradiol (E2) treatment in animals protects against acoustic trauma and aminoglycoside toxicity and its depletion can contribute to age-related hearing loss. However, estradiol is not suitable in clinical practice, given the increased risk of cancer and thromboembolic events. There is a need to find new treatments that present a higher benefit/risk ratio. In this work, we studied a new natural estrogen derivative, estetrol (E4), that was shown to act as a Selective Estrogen Receptor Modulator (SERM). Estetrol is thus able to display estrogen-like as well as antiestrogen activity depending on the target tissue. Interestingly, E4 allows the prevention of menopause symptoms such as osteoporosis or hot flushes through an agonistic action, whereas it exerts anti-estrogenic effects on the mammary gland. These preclinical data suggest that E4 presents a safer profile than E2. Material and Methods: To investigate whether this molecule presents otoprotective actions, we exposed P2 cultured organs of Corti to gentamicin, with or without estetrol (E4). After 48 hours of culture, hair cell survival was evaluated following anti-myosin VII Immunohistochemistry. We next moved on to in vivo studies using young zebrafish larvae (5dpf), since they constitute a highly effective model to determine the potential ototoxicity of drugs and to screen for candidates of hair cell protective agents. We monitored the survival of hair cells within neuromasts following chronic or acute exposure to gentamicin. Results: In cochlear culture, gentamicin-induced hair cell loss was significantly reduced by E4. Interestingly, the toxicity of aminoglycoside was also reduced in vivo upon treatment of zebrafish larvae with E4. Conclusion: The survival rate of hair cells was increased in the presence of estetrol both in vitro and in vivo indicating that estetrol constitutes an interesting protective drug against hearing loss. [less ▲]

The Elongator complex is required for proper development of the cerebral cortex. Interfering with its activity in vivo delays the migration of postmitotic projection neurons, at least through a defective ... [more ▼]

The Elongator complex is required for proper development of the cerebral cortex. Interfering with its activity in vivo delays the migration of postmitotic projection neurons, at least through a defective alpha-tubulin acetylation. However, this complex is already expressed by cortical progenitors where it may regulate the early steps of migration by targeting additional proteins. Here we report that connexin-43 (Cx43), which is strongly expressed by cortical progenitors and whose depletion impairs projection neuron migration, requires Elongator expression for its proper acetylation. Indeed, we show that Cx43 acetylation is reduced in the cortex of Elp3cKO embryos, as well as in a neuroblastoma cell line depleted of Elp1 expression, suggesting that Cx43 acetylation requires Elongator in different cellular contexts. Moreover, we show that histones deacetylase 6 (HDAC6) is a deacetylase of Cx43. Finally, we report that acetylation of Cx43 regulates its membrane distribution in apical progenitors of the cerebral cortex. [less ▲]

Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors with increased bioavailability, especially benfotiamine, exert neuroprotective effects not only for thiamine deficiency (TD), but also in mouse models of neurodegeneration. As it is known that AHN is impaired by stress in rodents, we exposed C57BL6/J mice to predator stress for 5 consecutive nights and studied the proliferation (number of Ki67-positive cells) and survival (number of BrdU-positive cells) of newborn immature neurons in the subgranular zone of the dentate gyrus. In stressed mice, the number of Ki67- and BrdU-positive cells was reduced compared to non-stressed animals. This reduction was prevented when the mice were treated (200 mg/kg/day in drinking water for 20 days) with thiamine or benfotiamine, that were recently found to prevent stress-induced behavioral changes and glycogen synthase kinase-3β (GSK-3β) upregulation in the CNS. Moreover, we show that thiamine and benfotiamine counteract stress-induced bodyweight loss and suppress stress-induced anxiety-like behavior. Both treatments induced a modest increase in the brain content of free thiamine while the level of thiamine diphosphate (ThDP) remained unchanged, suggesting that the beneficial effects observed are not linked to the role of this coenzyme in energy metabolism. Predator stress increased hippocampal protein carbonylation, an indicator of oxidative stress. This effect was antagonized by both thiamine and benfotiamine. Moreover, using cultured mouse neuroblastoma cells, we show that in particular benfotiamine protects against paraquat-induced oxidative stress. We therefore hypothesize that thiamine compounds may act by boosting anti-oxidant cellular defenses, by a mechanism that still remains to be unveiled. Our study demonstrates, for the first time, that thiamine and benfotiamine prevent stress-induced inhibition of hippocampal neurogenesis and accompanying physiological changes. The present data suggest that thiamine precursors with high bioavailability might be useful as a complementary therapy in several neuropsychiatric disorders. [less ▲]

It is commonly assumed that mammalian cochlear cells do not regenerate. Therefore, if hair cells are lost following an injury, no recovery could occur. However, during the first postnatal week, mice ... [more ▼]

It is commonly assumed that mammalian cochlear cells do not regenerate. Therefore, if hair cells are lost following an injury, no recovery could occur. However, during the first postnatal week, mice harbor some progenitor cells that retain the ability to give rise to new hair cells. These progenitor cells are in fact supporting cells. Upon hair cells loss, those cells are able to generate new hair cells both by direct transdifferentiation or following cell cycle re-entry and differentiation. However, this property of supporting cells is progressively lost after birth. Here, we review the molecular mechanisms that are involved in mammalian hair cell development and regeneration. Manipulating pathways used during development constitute good candidates for inducing hair cell regeneration after injury. Despite these promising studies, there is still no evidence for a recovery following hair cells loss in adult mammals. Stem Cells 2017. [less ▲]

Alström Syndrome (AS) is a human autosomal recessive genetic disorder characterized by numerous clinical symptoms including deafness. AS is caused by mutations in the ALMS1 gene encoding for ALMS1 protein ... [more ▼]

Alström Syndrome (AS) is a human autosomal recessive genetic disorder characterized by numerous clinical symptoms including deafness. AS is caused by mutations in the ALMS1 gene encoding for ALMS1 protein expressed at the basal body and implicated in ciliogenesis, cell cycle and proliferation (Jagger et al., 2011; Zulato et al., 2011 & Shenje et al., 2014). We are interesting in understanding the unknown mechanisms involving this protein in the genetic deafness of AS patients. To develop a model as closer as possible to the human pathology, we are using human induced pluripotent stem cells (hiPSCs) generated from fibroblasts of healthy and AS patients. Using a stepwise protocol, we demonstrated that healthy hiPSCs (waiting for isogenic hiPSCs) can generate a population of cells with gene and protein expression patterns consistent with the ones of otic progenitor cells (OSCs). At this differentiation stage, we observed some proliferation and apoptotic defects between healthy and AS cells. When human OSCs are co-cultured with mouse feeder cells, they are able to differentiate into hair cells (HCs). We successfully differentiated AS hiPSCs generated from AS patients into HCs. We are currently confirming gene expression pattern and testing HCs functionality. To exclude patient linked epigenetics and differentiation defects, we are correcting the genomic mutation in the AS hiPSCs to generate isogenic hiPSCs using the CRIPSR/Cas9 system. Thanks to the isogenic hiPSCs we will be able to confirm that these defects are well due to the ALMS1 mutation. [less ▲]

Elp3 lysine acetyl-transferase, the catalytic subunit of the Elongator complex, has been assigned multiple roles in gene transcription, DNA methylation and protein translation efficiency. Given the ... [more ▼]

Elp3 lysine acetyl-transferase, the catalytic subunit of the Elongator complex, has been assigned multiple roles in gene transcription, DNA methylation and protein translation efficiency. Given the importance of acetylation homeostasis in controlling developmental processes together with recent reports implicating Elp3 in cortical neurogenesis, we investigated its role during inner ear formation. In the inner ear, we detected Elp3 transcript in the sensory epithelia of the entire otic vesicle at embryonic day E11.5. At later stages, Elp3 mRNA is strongly expressed in the vestibular and spiral ganglion neurons. To investigate the role of Elp3 in vivo, we used a conditional knock-out mice (Foxg1Cre) in which the expression of the acetyl-transferase is lost in early otocyst. These mice show obvious vestibular defects as indicated by a stereotyped circling ambulation, head bobbing, retropulsion and the absence of a reaching response in the tail-hanging test. Furthermore, we identified a severe hearing loss in Elp3cKO mice through Auditory Brainstem Responses. We show that Elp3 enzyme is crucial for neuronal survival in the spiral ganglion and in the vestibule and that it ensures a correct innervation pattern in the developing inner ear. In the absence of Elp3, a drastic increase in the number of apoptotic neurons was detected by active Caspase-3 and pH2AX immunostainings, particularly during the early stages of development (between E12.5 and E14.5). Postnatally, the neurons remaining in Elp3cKO cochleae seem to establish synaptic contacts with the sensory cells but show obvious signs of cell damage as evidenced by Transmission Electron Microscopy. Taken together, these data support a role for Elp3 in hearing and balance and point out an important role for acetylation homeostasis during inner ear formation. We are currently investigating the molecular mechanisms underlying Elp3 effect on neuronal survival and pathfinding. [less ▲]

We planned to investigate the role of Elp3 acetyl-transferase, a member of the Elongator complex, in inner ear formation. We first analysed the spatio-temporal pattern of Elp3 mRNA expression and showed ... [more ▼]

We planned to investigate the role of Elp3 acetyl-transferase, a member of the Elongator complex, in inner ear formation. We first analysed the spatio-temporal pattern of Elp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea, in the spiral ganglion and in the vestibule. To unravel functions of Elp3, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3cKO). We submitted these mice to a battery of vestibular testing and found significant abnormalities. Besides, the auditory brain stem response of Elp3cKO indicated that these mice are severely deaf. We were also able to demonstrate an increased level of apoptosis in the Elp3cKO spiral ganglion leading to a reduced number of neurons and fibers innervating the sensory cells as well as a reduced number of their synaptic ribbons. Moreover, the remaining spiral ganglion neurons extend processes showing clearly defects regarding sensory cell innervation. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson's disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of ... [more ▼]

Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson's disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of this mutation and its possible developmental implications, we developed an in vitro assay to model PD with human induced pluripotent stem cells (hiPSCs) reprogrammed from skin fibroblasts of PD patients suffering from the LRKK2 G2019S mutation. We differentiated the hiPSCs into neural stem cells (NSCs) and further into dopaminergic neurons. Here we show that NSCs bearing the mutation tend to differentiate less efficiently into dopaminergic neurons and that the latter exhibit significant branching defects as compared to their controls. [less ▲]

The migration of cortical interneurons is a fundamental process for the establishment of cortical connectivity and its impairment underlies several neurological disorders. During development, these ... [more ▼]

The migration of cortical interneurons is a fundamental process for the establishment of cortical connectivity and its impairment underlies several neurological disorders. During development, these neurons are born in the ganglionic eminences and they migrate tangentially to populate the cortical layers. This process relies on various morphological changes that are driven by dynamic cytoskeleton remodelings. By coupling time lapse imaging with molecular analyses, we show that the Elongator complex controls cortical interneuron migration in mouse embryos by regulating nucleokinesis and branching dynamics. At the molecular level, Elongator fine-tunes actomyosin forces by regulating the distribution and turnover of actin microfilaments during cell migration. Thus, we demonstrate that Elongator cell-autonomously promotes cortical interneuron migration by controlling actin cytoskeletal dynamics.Cell Research advance online publication 27 September 2016; doi:10.1038/cr.2016.112. [less ▲]

The auditory organ is a highly specialized structure composed by specific cellular types. The sensory cells are characterized by stereocilia at their apex and are necessary for the sound perception. These ... [more ▼]

The auditory organ is a highly specialized structure composed by specific cellular types. The sensory cells are characterized by stereocilia at their apex and are necessary for the sound perception. These cells are supported by supporting cells which contain up to 3000 microtubules and are formed by 15protofilaments (15pf) instead of the canonical 13, a unique fact among vertebrates. It was shown that a β-tubulin isoform can specify the microtubule architecture, such as the expression of the Moth β2 in the Drosophila testes imposed the 16pf structure on the corresponding subset of Drosophila microtubules, which normally contain 13pf. To determine the role of these tubulins in the auditory organ and their possible involvement in the formation of the unusual 15pf microtubules of supporting cells, we studied the spatiotemporal localization of five -tubulin isotypes (β1 to 5) as well as acetyl-α-tubulin within the hearing organ during development in rodents. By using confocal microscopy, we showed that with the exception of the β3-tubulin isoform that was specific to nerve fibres, all the different β-tubulin isoforms and acetyl-α-tubulin were mainly present in the supporting cells. Contrary to β1-4-tubulins, we also found that the β5-tubulin isoform appeared only at a key stage of the postnatal development. By using transmission electron microscopy, we examined the fine structure of microtubules at an early and a late postnatal stage. Our TEM study indicated that these cells are composed by 13pf microtubules at P2, but by 15pf microtubules at P25. We revealed further that this developmental stage coincide with the formation of two separate bundles of microtubules from a unique one in these supporting cells. In conclusion, the architecture and composition of microtubules present in the supporting cells change during development of the organ of Corti. Further experiments are now required to determine if these changes are related to the appearance of β5-tubulin. [less ▲]

Post-translational modifications (PTMs) are key molecular events that modify proteins after their synthesis and modulate their ultimate functional properties by affecting their stability, localisation, interaction potential or activity. These chemical changes expand the size of the proteome adding diversity to the molecular pathways governing the biological outcome of cells. PTMs are thus crucial in regulating a variety of cellular processes such as apoptosis, proliferation and differentiation and have been shown to be instrumental during embryonic development. In addition, alterations in protein PTMs have been implicated in the pathogenesis of many human diseases, including deafness. In this review, we summarize the recent progress made in understanding the roles of PTMs during cochlear development, with particular emphasis on the enzymes driving protein phosphorylation, acetylation, methylation, glycosylation, ubiquitination and SUMOylation. We also discuss how these enzymes may contribute to hearing impairment and deafness. [less ▲]

We planned to investigate the role of Elp3 acetyl-transferase, a member of the Elongator complex, in inner ear formation. We first analysed the spatio-temporal pattern of Elp3 mRNA expression and showed ... [more ▼]

We planned to investigate the role of Elp3 acetyl-transferase, a member of the Elongator complex, in inner ear formation. We first analysed the spatio-temporal pattern of Elp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea, in the spiral ganglion and in the vestibule. To unravel functions of Elp3, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3cKO). We submitted these mice to a battery of vestibular testing and found significant abnormalities. Besides, the auditory brain stem response of Elp3cKO indicated that these mice are severely deaf. We were also able to demonstrate an increased level of apoptosis in the Elp3cKO spiral ganglion leading to a reduced number of neurons and fibers innervating the sensory cells as well as a reduced number of their synaptic ribbons. Moreover, the remaining spiral ganglion neurons extend processes showing clearly defects regarding sensory cell innervation. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine. [less ▲]

Given the importance of acetylation homeostasis in controlling developmental processes [1-3], we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase ... [more ▼]

Given the importance of acetylation homeostasis in controlling developmental processes [1-3], we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis [4]. To determine the role of Elp3 in the inner ear, we first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of neurons and fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons at P15. Moreover, the remaining spiral ganglion neurons extend processes showing clearly defects regarding hair cells innervation (misorientation of fibers). In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

The inner ear is a complex organ composed of the vestibular system – which is the balancing system – and the cochlea – which is the earing system. The cochlea is a coiled shape organ composed of three ... [more ▼]

The inner ear is a complex organ composed of the vestibular system – which is the balancing system – and the cochlea – which is the earing system. The cochlea is a coiled shape organ composed of three main structures: the spiral ligament sitting on top of the stria vascularis, the organ of Corti with sensory hair cells and supporting cells and the spiral ganglion composed of neurons and glial cells. After an auditory stimulus, the sound wave progresses in the scala media filled with endolymph and induces a stimulation of sensory hair cells. These cells then transmit the information to the spiral ganglion neurons connected to them. Of course, the correct ionic homeostasis of endolymph is required for a good sound wave transmission. This homeostatic function is assured by the stria vascularis and the spiral ligament. The alteration of one of the structures mentioned before induces deafness. Currently, numerous genes have been associated to this kind of hearing loss. In the present work, we focus our attention Cemip – also known as KIAA1199 – that has been associated to human hereditary neurosensory deafness. Indeed, three missense mutations consisting in non-synonymous amino acid changes (R187L, R187H and H783Y) have been associated to this form of deafness. Therefore we would like to understand the role of Cemip in the cochlea. For that we have analysed Cemip mRNA pattern of expression by in situ hybridization at different developmental stages on cochlear sections. It seems Cemip mRNA is not present in the auditory portion of the inner ear at early embryonic stage 14 (E14) while it is largely present at E17 in the spiral ganglion, in supporting cells of the organ of Corti and in the spiral ligament. This expression is maintained post-nattily until P7. At P21 the expression is restricted to the spiral lamina - an osseous structure surrounding the spiral ganglion. Our on going work is aimed at revealing the biological role of Cemip in the cochlea in conditional knock-out mice. [less ▲]

In mammals, cochlear sensory hair cells that are responsible for hearing are postmitotic and are not replaced after loss. One of the most promising strategies to regenerate hair cells is to identify and ... [more ▼]

In mammals, cochlear sensory hair cells that are responsible for hearing are postmitotic and are not replaced after loss. One of the most promising strategies to regenerate hair cells is to identify and inhibit the factors preventing the conversion of adjacent non-sensory supporting cells into hair cells. Here we demonstrate that mammalian hair cells can be directly generated from supporting cells by inhibition of ephrin-B2 signalling. Using either ephrin-B2 conditional knockout mice, shRNA-mediated gene silencing or soluble inhibitors, we found that downregulation of ephrin-B2 signalling at embryonic stages results in supporting cell translocation into hair cell layers and subsequent switch in cell identity from supporting cell to hair cell fate. As transdifferentiation is here a result of displacement across boundary, this original finding presents the interest that newly generated hair cells directly integrate either hair cell layer, then would be likely more rapidly able to fit into functional circuitry. [less ▲]